Director PCR

Product description The kit is equipped with a powerful lysis buffer, which can quickly lyse samples (e.g. mouse tail, mouse ear, mouse toe, muscle, etc.) to release genomic DNA, and the lysate can be directly added to the PCR reaction system without purification, which is convenient for operation. In addition, this kit requires low sample input, 5 mg of mouse tissue or 1-5 mm of mouse tail can be used for experiments. Components Components No. Name N132028E (50 T) N132028S  (200 T) N132028-A Buffer ML 5×1 mL 20 mL N132028-B Buffer MT 0.6 mL 2×1.25 mL 1) Buffer ML is a lysis buffer containing strong protein denaturants, please wear gloves. 2) Buffer MT is a stop buffer used to stop the lysis function of Buffer ML. Storage 1. Component A: The product should be stored at 2-8℃ for one year. For multiple use for a long time, please avoid cross-contamination. 2. Component B: The product should be stored at -25 ~ -15℃ for one year. Please avoid repeated freeze-thaw. Notes 1. This product is for research use only. 2. Please operate with lab coats and disposable gloves，for your safety. 3. In order to avoid cross contamination between samples, it is necessary to immerse the edge of the sampling device or the part in direct contact with the sample in 2% sodium hypochlorite solution after each sampling, wash it several times, and then dry the residual liquid with a clean paper towel before using it again. For the convenience of the test, several sampling devices can be prepared and cleaned uniformly after use to ensure that each individual sample is sampled with a contamination-free sampling device. 4. It is recommended to use freshly taken animal tissues. In case of long-term frozen tissues, repeated freezing and thawing should be avoided as much as possible, otherwise it will lead to degradation of the template and affect the PCR efficiency. Instructions Mouse Genomic DNA Release 1. Clip 5-10 mg of animal tissue or 1-5 mm mouse tail in a 1.5 mL centrifuge tube*. 【Note】: * Tissue should be cut up as much as possible to allow the lysis reaction to proceed more smoothly. 2. Add 90 μL of Buffer ML to the above centrifuge tube, vortex gently to completely infiltrate the sample with the lysate, and centrifuge briefly. 3. Incubate at 95°C for 15 min in a thermostatic incubator**. 【Note】: **Incubation at 95°C for 15 min is generally sufficient for most PCR needs. If a larger amount of DNA is required or the sample is difficult to lyse, the time can be extended to 30 min. The tissue block does not need to be completely lysed, and the residue can be removed in a subsequent centrifugation step. 4. Add 10 μL of Buffer MT, flick to mix, and terminate lysis. 5. Optional step: centrifugation at 12000 rpm for 2 min. 6. Transfer the supernatant to a new centrifuge tube and store at 4°C or -20°C or take the supernatant directly for subsequent PCR amplification. Cell Genomic DNA Release After culturing transfected cells in a 48-well-plate for 3 days and reaching a cell density of about 70,000 cells/well, remove the medium as completely as possible. Then add 90 μL of ML buffer directly per well and pipette each well. Transfer all to the 96 well-PCR plate. After treating with a PCR machine at 95 °C for 5-15 min and cooling down, add 10 μL MT buffer and blow evenly and thoroughly. Using high-fidelity enzyme (Cat# N132016) or Taq enzyme, add 0.5-2 μL cell lysate to every 50 uL of PCR reaction system and perform PCR.

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Product description Plant tissue direct PCR kit is a kit that can directly amplify different types of plant leaves by PCR, with wide adaptability and strong stability. The kit uses a unique lysis buffer system, which can quickly lyse a variety of plant samples and release genomic DNA. The released genomic DNA can be used directly as a template without removing protein, RNA, or secondary metabolites in PCR reaction. In addition, the kit requires a small amount of sample, as low as 1 mm plant leaves can be used for experiments. The 2×Plant Master Mix provided in this kit has strong amplification compatibility and can directly use the lysate of the sample as a template for efficient and specific amplification. This reagent is a 2-fold concentrated PCR reaction mixture, which contains all the components used for PCR amplification except the template and primers, which greatly simplifies the operation process and reduces the chance of contamination. The kit can be used for identification of transgenic plants, plant genotyping, etc. Components Components No. Name N132023E (50 T) N132023S  (200 T) Storage N132023-A Buffer P1 2×1.25 mL 2×5 mL 2-8℃ N132023-B Buffer P2 500 μL 2×1 mL 2-8℃ N132023-C 2×Plant Master Mix* 500 μL 2×1 mL -25 ~ -15℃ 【Note】: *2×Plant Master Mix: Contains hot-start Taq DNA polymerase, dNTP mix, MgCl2, reaction buffer, PCR reaction enhancer, optimizer and stabilizer, etc. It also includes electrophoresis Loading Buffer, which can be directly used in electrophoresis after PCR. Shipping and Storage 1. Component A: The product should be stored at 2-8℃ for one year. For multiple use for a long time. 2. Component B: The product should be stored at 2-8℃ for one year. It is used for neutralizing lysate, which is beneficial to store the sample for a longer time. 3. Component C: The product should be stored at -25 ~ -15℃ for one year. Please avoid repeated freeze-thaw. Notes 1) When doing leaf experiments, it is recommended to use freshly collected leaf tissue. If it is a long-term frozen tissue, it needs to be stored at -80℃. Repeated freezing and thawing should be avoided as much as possible to avoid template degradation and affect PCR efficiency. The leaf tissue is suitable for young leaves. If it is a mature leaf, avoid using the tissue of the main vein of the leaf. 2) It is recommended to amplify the fragment within 1 kb in length for the best amplification efficiency. 3) When sampling, use a hole punch or knife to take a sample of suitable size. When the samples are different, the hole punch or knife needs to be cleaned every time before processing the sample. 4) For leaf tissue, it is recommended to take 1-10 mm leaves, too small leaf length will lead to low PCR amplification yield, too much will inhibit the PCR reaction, use the method of thermal cracking, mashing with a pipette tip, and crushing with a grinder to treat plant leaves. After treatment, it needs to be shaken and centrifuged. Be sure to take the supernatant for testing. Precipitation will seriously inhibit the PCR reaction. 5) For your safety and health, please wear lab coats and disposable gloves for operation. 6) This product is for research use ONLY! Instructions 1. Plant leaves 1.1 Grinding and cracking method: 1) Crushing with a grinder: Put the leaves with a diameter of about 5 mm in 50 μL Buffer P1, add steel beads (about 3 mm in diameter, 2 in total) in grinder to break the leaves (45 Hz, 1 min), and the solution with broken leaves will appear green. Centrifuge briefly, keep the supernatant at 4℃ for later use, and take 1 μL for PCR amplification. 2) Mashing with pipette tip: It is recommended to use young leaves. Place leaves with a diameter of about 5 mm in 50 μL Buffer P1, mash the leaves with a pipette tip, the solution turns green after mashing. Centrifuge briefly, keep the supernatant at 4℃ for later use, and take 1 μL for PCR amplification. 1.2 Thermal cracking method: It is recommended to use young leaves. Place leaves with a diameter of about 5 mm in 50 μL Buffer P1 and heat at 95°C for 5-10 min (to ensure that the lysate is completely submerged in the leaves), for leaves that are difficult to lyse (old leaves), the time can be appropriately extended (10-20 min). The solution turns green after heating and lysing. Shake and mix well, centrifuge briefly, keep the supernatant at 4℃ for later use, and take 1 μL for PCR amplification. 1.3 Direct method: It is recommended to use young leaves. Use a hole punch or a knife to directly add leaves with a diameter of about 1 mm into the PCR reaction system; for complex samples or amplification of long fragments, it is recommended to use leaves with a diameter of less than 1 mm. 2. Reaction System Table 1 Amplification system Components Volume (μL) Volume (μL) Final concentration 2× Plant Master Mix 10 25 1× Forward Primer (10 μmol/L) 0.5 1 0.2-0.4 μmol/L Reverse Primer (10 μmol/L) 0.5 1 0.2-0.4 μmol/L Cleavage product (DNA template) 1 2 - ddH2O to 20 to 50 - 【Note】: *All components should be thoroughly mixed before use. a. The amount of template added: less than 5% of the PCR reaction system. Excessive amount will seriously inhibit the PCR reaction. It is strongly recommended to add 1 μL template. For direct amplification of leaves, the grinder cracking method is preferred. b. Final primer concentration: Using 0.2-0.25 μmol/L primer to get better results. When the reaction performance is poor, the primer concentration can be adjusted in the range of 0.1-0.5 μmol/L. c. Reaction system: 20 μL or 50 μL is recommended to ensure the validity and repeatability of target gene amplification. d. System preparation: Prepare the PCR reaction system, place it on a vortexer, vortex and mix, and centrifuge briefly to collect the reaction solution at the bottom of the tube. e. Control reaction: It is recommended to set positive and negative PCR control reactions in order to eliminate the interference of false positives or false negatives when performing PCR. f. In order to store the lysed template more stably, mix the transferred supernatant according to the ratio of lysis product (DNA template): Buffer P2 = 5:1, and store at -20℃ after mixing. Stable preservation varies with time and sample status. If the treated plant leaf supernatant is used for PCR amplification within one week, without adding Buffer P2, the supernatant should be stored at -20℃. 3. Reaction Program             Table 2 Amplification protocol Cycle steps Temperature (℃) Time Cycles Predenaturation 94 5 min 1 Denaturation 94 10 sec 35 Annealing 50-65 20 sec Extension 72 1 min Final extension 72 5 min 1 【Note】： 1) Annealing temperature: Please refer to the theoretical Tm value of the primer. The annealing temperature can be set 2-5℃ lower than the theoretical value of the primer. 2) Extension time: It needs to be determined according to the length of the fragment. For DNA fragments within 1 kb, the recommended extension time is 1 min.  

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Product description This Kit is a kit that enables direct PCR amplification of whole blood samples without DNA   purification or sample pretreatment. This kit is compatible with fresh blood containing conventional anticoagulants such as EDTA, heparin, citrate, frozen blood and commercial Whatman903™ and FTA™ dried blood stains. The kit contains a combination of genetically engineered DNA Polymerase with high fidelity and tolerance to PCR inhibitors. It can efficiently amplify 8 kb genomic fragments under rapid elongation conditions, for the fragments less than 2  kb, the extension time of 3-5 sec/kb can be set to complete the amplification, thus significantly reducing the identification and detection time of blood samples. Advanced High-Fidelity DNA Polymerase Mix has a flat 3 'end of the amplified product, which is suitable for one-step rapid cloning (CAT#N312081) and TOPO cloning. The Positive control primer mix (10 μmol/L each) provided in the kit is capable of amplifying a fragment 237 bp in length from the upstream conserved sequence of the sox21 gene in mammals and most vertebrates, which could be used as a positive control. Components Components No. Name N132022E (50 T) N132022S (100 T) N132022-A 2× Blood Advanced PCR Buffer 1.25 mL 2.5 mL N132022-B Advanced High-Fidelity DNA Polymerase Mix 50 μL 100 μL N132022-C Positive control primer mix (10 mol/L each) 100 μL 200 μL N132022-D 10×DNA loading buffer 1 mL 1 mL Shipping and Storage The product should be stored at -25 ~ -15℃ for one year. Please avoid repeated freeze-thaw. Notes 1. The recommended amount of blood template is 10% of the total reaction volume, i.e. 5 μL of whole blood in 50 μL reaction system as a template, taking care to avoid suction of blood clots. 2. Most of the target fragments below 8 kb could be amplified with the extension time of 10 sec/kb, and most of the fragments below 2 kb could be amplified with the extension time of     3-5 sec/kb. If the amplification efficiency is low, it can be used when extended to 30 sec/kb. 3. After PCR reaction, it is recommended to centrifuge the reaction product at 4000 rpm (1000 x g) for 1-3 min to precipitate blood cell debris and remove the supernatant for downstream analysis. 4. This product should not be used directly for medical diagnosis. 5. For your safety and health, please wear lab coats and disposable gloves for operation. 6. This product is for research use ONLY! Instructions 1. Reaction System Table 1 Amplification protocol Components Volume (μL) 2× Blood Advanced PCR Buffer 25 Forward Primer (10 μmol/L) 2 Reverse Primer (10 μmol/L) 2 Advanced High-Fidelity DNA Polymerase Mix 1 ddH2O to 50 【Note】: *All components should be thoroughly mixed before use. 1) Primer final concentration: The recommended final concentration for each primer is 0.4   μmol/L, too high will result in non-specific amplification. 2) Template usage: The optimal whole blood template concentration range is 0.5%-20%, and the recommended dosage is 10% as the initial trial condition, i.e. 5 μL whole blood in 50 μL reaction system as a template, taking care to avoid suction of blood clots. For dried blood stains stored on WhatmanTM filter paper cards, a round paper of about 1 mm2 with blood stains can be used for amplification without pretreatment and directly placed into the PCR reaction solution. 2. Reaction Program Cycle steps Temperature (℃) Time Cycles Predenaturation 95 5 min 1 Denaturation 95 15 sec 30-35 Annealing 60 15 sec Extension 72 3-10 sec/kb Final extension 72 5 min 1   【Note】: a. Annealing temperature: please  refer to the theoretical Tm value of the primer or 1-2°C below the primer Tm value. If amplification product specificity is poor, an annealing  temperature gradient can be established to find optimal annealing conditions. b. Extension time: Most of the target fragments below 8 kb could be amplified with the extension time of 10 sec/kb, and most of the fragments below 2 kb could be amplified with the extension time of 3-5 sec/kb. If the amplification efficiency is low, the time can be appropriately extended to 20-30 sec/kb and should not exceed 60 sec/kb.

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Product description This kit can directly and quickly conduct PCR amplification of mouse tissue (such as mouse tail, mouse ear, mouse toe, muscle, etc.), and has strong sample compatibility. Equipped with a powerful lysis buffer, this kit can rapidly lyse samples and release genomic DNA. The lysate can be directly added to the PCR reaction system without purification, and the operation is convenient. In addition, this kit requires low sample input, and 5 mg mouse tissue or 1-5 mm mouse tail can be used for experiments. The 2×Mouse Direct PCR Mix provided by this kit is a hot-start PCR reaction solution with a 2-fold concentration. It contains all the components used for PCR amplification except the template and primers, which greatly simplifies the operation process and reduces the chance of contamination. The kit can be used for transgene identification, mouse genotyping, etc. Components Components No. Name N132021E (50 T) N132021M (200 T) N132021-A Buffer ML 5×1 mL 20×1 mL N132021-B Buffer MT 0.6 mL 2×1.25 mL N132021-C 2×Mouse Direct PCR Mix 500 μL 2×1 mL 【Note】: 1) Buffer ML is a lysis buffer containing strong protein denaturants, please wear gloves. 2) Buffer MT is a stop buffer used to stop the lysis function of Buffer ML. 3) 2×Mouse Direct PCR Mix: Contains hot-start Taq DNA polymerase, dNTP mix, MgCl2, reaction buffer, PCR reaction enhancer, optimizer, stabilizer, electrophoresis indicator dye, etc. Specifications Product specification Kit Hot Start Built-in Hot Start Overhang Blunt Conditions for transportation Ice Packs Product Type Direct PCR Kit Apply to (application) Mouse tail, Mouse ear, Rat toe, Viscera, Skin, etc. Storage 1. Component A: The product should be stored at 2-8℃ for one year. For multiple use for a long time, please avoid cross-contamination. 2. Component B/C: The product should be stored at -25 ~ -15℃ for one year. Please avoid repeated freeze-thaw. Applications This product is primarily applied to the genotyping of mice. Notes 1. To avoid sample to sample cross contamination, the edge of the sampling equipment or the site in direct  contact with the sample was immersed in 2% sodium hypochlorite solution after each sampling session,  washed repeatedly several times, and then blotted with clean paper towels to dry the residual liquid before use. For the convenience of the test, multiple sampling equipment can also be prepared and cleaned uniformly after use to ensure that each individual sample uses a non-polluting sampling equipment. 2. It is recommended to use freshly collected animal tissue. If it is a long-term frozen tissue, repeated freezing and thawing should be avoided as much as possible, otherwise the template will be degraded and the PCR efficiency will be affected. 3. It is recommended to amplify the fragment length within 1 kb for the best amplification efficiency. 4. For your safety and health, please wear lab coats and disposable gloves for operation. 5. This product is for research use ONLY! Instructions 1. Sample genomic DNA release 1.1 Cut 5-10 mg of animal tissue or 1-5 mm of rat tail and put them in a 1.5 mL centrifuge tube. 1.2 Add 90 μL of Buffer ML to the above centrifuge tube, vortex gently so that the sample is completely infiltrated by the lysate, and centrifuge briefly. 1.3 Incubate at 95℃ for 15 min in a constant temperature incubator. 1.4 Add 10 μL Buffer MT, flick and mix to stop lysis. 1.5 Optional steps: centrifugation at 12,000 rpm for 2 min. 1.6 Transfer the supernatant to a new centrifuge tube and store at 4℃ or -20℃ or directly take the supernatant for subsequent PCR amplification. 【Note】: *The tissue should be chopped as little as possible to allow the lysis reaction to proceed more smoothly. **Incubate at 95℃, usually 15 min to meet most PCR needs. If a larger amount of DNA is required or the sample is difficult to lyse, the time can be extended to 30 min. The tissue mass does not need to be completely lysed. The residual fraction can be removed in a subsequent centrifugation step. 2. Reaction System Table 1  Reaction system Components Volume (μL) Final concentration 2×Mouse Direct PCR Mix 10 1× Forward Primer (10 μmol/L) 0.5 0.2-0.4 μmol/L Reverse Primer (10 μmol/L) 0.5 0.2-0.4 μmol/L Lysate product (DNA template) 1 - ddH2O to 20 - *All components should be thoroughly mixed before use. 2.1 Template usage: It is recommended to use 1-10% of the total system amount of template, and 1 μL supernatant is recommended as template for 20 μL system. 2.2 Primer final concentration: it is recommended to use 0.2-0.4 μmol/L of primer final concentration to get better results. When the reaction performance is poor, the primer concentration can be adjusted in the range of 0.1-0.5 μmol/L. 2.3 Reaction system: 20 μL is recommended, and the volume of the system can also be adjusted according to usage habits. 2.4 System preparation: Prepare the PCR reaction system, place it on a vortexer, vortex and mix, and centrifuge briefly to collect the reaction solution at the bottom of the tube. 3. Reaction Program Table 2  Reaction Program Cycle steps Temperature (°C) Time Cycles Predenaturation 94 5 min 1 Denaturation 94 10 sec 35 Annealing 60 20 sec Extension 72 30 sec/kb Final extension 72 5 min 1 3.1 Annealing temperature: please refer to the theoretical Tm value of the primer. The annealing temperature can be set 2-5℃ lower than the theoretical value of the primer. 3.2 Extension time: please set as 30 sec/kb. 3.3 Number of amplification cycles: 35 cycles can amplify enough products. 3.4 Sample loading by electrophoresis: take 3-5 μL of amplification product and load it. 4.  Control response In the analysis of PCR results, whether positive or negative, the reliability of the results cannot be determined without a control reaction. In order to facilitate the analysis of subsequent experimental results, it is recommended to set positive and negative PCR control reactions during PCR in order to exclude false positive or false negative interference.

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